Pharmaceutical combinations comprising a histone deacetylase inhibitor and a cd38 inhibitor and methods of use thereof

ABSTRACT

The present disclosure relates to a pharmaceutical combination comprising (a) a histone deacetylase inhibitor and (b) a CD38 inhibitor, FIG. 1A including combined preparations and pharmaceutical compositions thereof; uses of such combination in the treatment or prevention of cancer; and methods of treating or preventing cancer in a subject comprising administering a therapeutically effective amount of such combination.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/425,980 filed Nov. 23, 2016, the contents of which are incorporatedherein by reference in their entirety.

BACKGROUND

Histone deacetylase (HDAC) inhibition can cause cancer cell growtharrest. However, pan-HDAC inhibition leads to significant adverseeffects, and an alternative HDAC inhibition profile is desirable.

HDAC6 is a class IIb HDAC and is known to remove acetyl groups from manycellular proteins, including α-tubulin and HSP90. It has been reportedthat HSP90 hyperacetylation destabilizes its target proteins, includingER and EGFR. Inhibitors of HDAC6 have demonstrated anti-cancerproliferative activity in various cancer types. Blocking HDAC6 activityhas been shown to cause cancer cell growth inhibition through variousmechanisms.

In spite of numerous treatment options for cancer patients, thereremains a need for effective and safe therapeutic options. Inparticular, there is a need for effective methods of treating orpreventing cancers, especially those cancers that have been resistantand/or refractive to current therapies. This need can be fulfilled bythe use of combination therapies such as those described herein.

SUMMARY

Provided herein is a pharmaceutical combination comprising a histonedeacetylase inhibitor (HDAC) inhibitor and a CD38 inhibitor.

In an aspect, provided herein is a pharmaceutical combination comprisinga therapeutically effective amount of a HDAC inhibitor, or apharmaceutically acceptable salt thereof, and a CD38 inhibitor.

In various embodiments, the combination further comprises a compoundselected from thalidomide or an analog thereof, or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the compound isselected from thalidomide, pomalidomide, and lenalidomide, or apharmaceutically acceptable salt thereof. In exemplary embodiments, thecompound is pomalidomide.

In various embodiments, the HDAC inhibitor is an HDAC6-selectiveinhibitor. In other embodiments, the HDAC inhibitor is an HDAC6inhibitor of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R₁ is aryl or heteroaryl, each of which may be optionally substituted byOH, halo, or C₁₋₆-alkyl;

and

R is H or C₁₋₆-alkyl.

In various embodiments of the pharmaceutical combination, ring B isaryl.

In various embodiments of the pharmaceutical combination, R₁ is aryl orheteroaryl, each of which may be optionally substituted by halo.

In various embodiments of the pharmaceutical combination, the HDAC6inhibitor of Formula I is:

or a pharmaceutical acceptable salt thereof.

In various embodiments of the pharmaceutical combination, the HDAC6inhibitor of Formula I is:

or a pharmaceutically acceptable salt thereof.

In various embodiments of the pharmaceutical combination, the CD38inhibitor is an inhibitory antibody. In an exemplary embodiment, theCD38 inhibitory antibody is daratumumab.

In various embodiments, the inhibitory CD38 antibody comprises a heavychain comprising an amino acid sequence within the amino acid sequenceof SEQ ID NO.: 1 or a portion thereof, and a light chain comprising anamino acid sequence within the amino acid sequence of SEQ ID NO.: 6 or aportion thereof. In another embodiment, the CD38 inhibitory antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO.: 2 or a portion thereof, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO.: 7 or aportion thereof. In various embodiments, the heavy chain comprises aderivative or a portion of the amino acid sequence of SEQ ID NO.: 1. Inother embodiments, the light chain comprises a derivative or a portionof the amino acid sequence of SEQ ID NO.: 6. In another embodiment, theheavy chain variable region comprises three CDRs comprising amino acidsequences of SEQ ID NOs.: 3-5 or a portion thereof. In anotherembodiment, the light chain variable region comprises three CDRscomprising amino acid sequences of SEQ ID NOs.: 8-10 or a portionthereof.

In various embodiments of the pharmaceutical combination, the HDAC6inhibitor and the CD38 inhibitor are in the same formulation. In otherembodiments of the pharmaceutical combination, the HDAC6 inhibitor andthe CD38 inhibitor are in separate formulations.

In various embodiments of the pharmaceutical combination, the HDACinhibitor is in a formulation for oral administration, and the CD38inhibitor is in a formulation for intravenous administration.

In various embodiments of the pharmaceutical combination, thepharmaceutical combination is used in the treatment of cancer in asubject in need thereof. In another embodiment, the pharmaceuticalcombination is used for the manufacture of a pharmaceutical preparationor medicament for the treatment of cancer.

In various embodiments of the pharmaceutical combination, the cancer isa hematologic cancer. In other various embodiments of the pharmaceuticalcombination, the cancer is selected from the group consisting ofmultiple myeloma, amyloidosis, plasma cell myeloma, smoldering myeloma,mantle-cell lymphoma, diffuse large B-cell lymphoma, follicularlymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, andHodgkin's lymphoma.

In an aspect, provided herein is a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of the pharmaceutical combinationprovided herein. In various embodiments of the method, the cancer is ahematologic cancer. In other various embodiments of the method, thecancer is selected from the group consisting of multiple myeloma,amyloidosis, plasma cell myeloma, smoldering myeloma, mantle-celllymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia,follicular lymphoma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

In an aspect, provided herein is a method for upregulatingantibody-dependent cell-mediated cytotoxicity in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical combination provided herein.

In an aspect, provided herein is a method for upregulating lymphocytefunctional activity in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of apharmaceutical combination provided herein. In various embodiments, thelymphocyte is a natural killer cell.

Other objects, features, and advantages will become apparent from thefollowing detailed description. The detailed description and specificexamples are given for illustration only because various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Further, the examples demonstrate the principle of the invention.

In various embodiments of the pharmaceutical combinations, thecombination further comprises dexamethasone.

In various embodiments of the methods, the method further comprisesadministering to the subject a therapeutically effective amount ofdexamethasone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are bar graphs showing the effect of Compound A andCompound B on surface expression levels of CD38 in RPMI-8226 (FIG. 1A)and MM.1S (FIG. 1B) multiple myeloma cells.

FIG. 2A and FIG. 2B are bar graphs showing the effect of increasingconcentrations of Compound A on anti-CD38 antibody-induced antibodydependent cell mediated cytotoxicity in H929 multiple myeloma cells.

FIG. 3A and FIG. 3B are bar graphs showing the effect of Compound B onanti-CD38 antibody-induced antibody dependent cell mediated cytotoxicityin patient-derived multiple myeloma cells in an autologous setting.Compound B alone was used as a control.

FIG. 4A is a line graph showing the effect of combination treatment ofCompound B and daratumumab on the suppression of tumor growth of Daudicell lymphomas. The lymphomas were treated with either (vehicle(diamond), IgG control (square), daratumumab alone (triangle), CompoundB and daratumumab (inverted triangle)). FIG. 4B is a line graph showingthe effect of combination treatment of Compound B and daratumumab on thesuppression of tumor growth of Daudi cell lymphomas (dashed) relative todaratumumab alone (solid), including the frequency of partial responses(PR) and complete responses (CR). FIG. 4C is a graph showing thefrequency of Daudi tumor regression after completion of dosing.

FIG. 5 is a bar graph showing the effect of pomalidomide on anti-CD38antibody-induced antibody dependent cell mediated cytotoxicity in H929multiple myeloma cells.

FIG. 6 is a is a bar graph showing the effect of Compound B incombination with pomalidomide on anti-CD38 antibody induced cellmediated cytotoxicity in H929 multiple myeloma cells.

DETAILED DESCRIPTION

Provided herein is a pharmaceutical combination comprising a histonedeacetylase inhibitor (HDAC) inhibitor and a CD38 inhibitor.

In an embodiment, the pharmaceutical combination comprises:

(a) a HDAC6 inhibitor of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R₁ is aryl or heteroaryl, each of which may be optionally substituted byOH, halo, or C₁₋₆-alkyl

and

R is H or C₁₋₆-alkyl; and

(b) a CD38 inhibitor, or a pharmaceutically acceptable salt thereof.

Definitions

Certain terms used herein are described below. Compounds of the presentinvention are described using standard nomenclature. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs.

The term “alkyl” refers to saturated, straight- or branched-chainhydrocarbon moieties containing, in certain embodiments, between one andsix, or one and eight carbon atoms, respectively. Examples of C₁₋₆-alkylmoieties include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties; andexamples of C₁₋₈-alkyl moieties include, but are not limited to, methyl,ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl,heptyl, and octyl moieties. The number of carbon atoms in an alkylsubstituent can be indicated by the prefix “C_(x-y),” where x is theminimum and y is the maximum number of carbon atoms in the substituent.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, chlorine.

The term “aryl” refers to a mono- or poly-cyclic carbocyclic ring systemhaving one or more aromatic rings, fused or non-fused, including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl,and the like. In some embodiments, aryl groups have 6 carbon atoms. Insome embodiments, aryl groups have from six to ten carbon atoms. In someembodiments, aryl groups have from six to sixteen carbon atoms. In anembodiment, C₅-C₇ aryl groups are provided herein.

The term “heteroaryl” refers to a mono- or poly-cyclic (e.g., bi-, ortri-cyclic or more) fused or non-fused moiety or ring system having atleast one aromatic ring, where one or more of the ring-forming atoms isa heteroatom such as oxygen, sulfur, or nitrogen. In some embodiments,the heteroaryl group has from about one to six carbon atoms, and infurther embodiments from one to fifteen carbon atoms. In someembodiments, the heteroaryl group contains five to sixteen ring atoms ofwhich one ring atom is selected from oxygen, sulfur, and nitrogen; zero,one, two, or three ring atoms are additional heteroatoms independentlyselected from oxygen, sulfur, and nitrogen; and the remaining ring atomsare carbon.

Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,quinoxalinyl, acridinyl, and the like. In an embodiment, C₄-C₇heteroaryl groups are provided herein.

As used herein, the term “pharmaceutically acceptable salt” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts of thepresent invention include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety.

The term “pharmaceutically acceptable” as used herein refers to thosecompounds, materials, compositions and/or dosage forms, which are,within the scope of sound medical judgment, suitable for contact withthe tissues a warm-blooded animal, e.g., a mammal or human, withoutexcessive toxicity, irritation allergic response and other problemcomplications commensurate with a reasonable benefit/risk ratio.

The terms “fixed combination,” “fixed dose,” and “same formulation” asused herein refers to a single carrier or vehicle or dosage formformulated to deliver an amount, which is jointly therapeuticallyeffective for the treatment of cancer, of both therapeutic agents to apatient. The single vehicle is designed to deliver an amount of each ofthe agents, along with any pharmaceutically acceptable carriers orexcipients. In some embodiments, the vehicle is a tablet, capsule, pill,or a patch. In other embodiments, the vehicle is a solution or asuspension.

The term “non-fixed combination,” “kit of parts,” and “separateformulations” means that the active ingredients, i.e., the HDAC6inhibitor and the CD38 inhibitor, are administered to a patient asseparate entities either simultaneously, concurrently or sequentiallywith no specific time limits, wherein such administration providestherapeutically effective levels of the two compounds in the body of thesubject in need thereof. The latter also applies to cocktail therapy,e.g., the administration of three or more active ingredients.

An “oral dosage form” includes a unit dosage form prescribed or intendedfor oral administration. In an embodiment of the pharmaceuticalcombinations provided herein, the HDAC6 inhibitor (e.g., Compounds A orB) is administered as an oral dosage form.

The term “treating” or “treatment” as used herein comprises a treatmentrelieving, reducing or alleviating at least one symptom in a subject oreffecting a delay of progression of a disease. For example, treatmentcan be the diminishment of one or several symptoms of a disorder orcomplete eradication of a disorder, such as cancer.

The term “prevent,” “preventing,” or “prevention” as used hereincomprises the prevention of at least one symptom associated with orcaused by the state, disease or disorder being prevented.

As used herein, the term “resistant” or “refractive” to a therapeuticagent when referring to a cancer patient means that the cancer hasinnate, or achieved resistance to, the effects of the therapeutic agentas a result of contact with the therapeutic agent. Stated alternatively,the cancer is resistant to the ordinary standard of care associated withthe particular therapeutic agent.

As used herein, the term “anti-CD38 naïve” refers to a patient or asubject or a cancer that has not previously been treated with ananti-CD38 antibody.

The term “pharmaceutically effective amount,” “therapeutically effectiveamount,” or “clinically effective amount” of a combination oftherapeutic agents is an amount sufficient to provide an observable orclinically significant improvement over the baseline clinicallyobservable signs and symptoms of the disorders treated with thecombination.

The term “jointly therapeutically active” or “joint therapeutic effect”as used herein means that the therapeutic agents can be given separately(in a chronologically staggered manner, especially a sequence-specificmanner) in such time intervals, in the warm-blooded animal, especiallyhuman, to be treated, still show an (preferably synergistic) interaction(joint therapeutic effect). Whether this is the case can, inter alia, bedetermined by following the blood levels of the compounds, showing thatboth compounds are present in the blood of the human to be treated atleast during certain time intervals.

The term “subject” or “patient” as used herein is intended to includeanimals, which are capable of suffering from or afflicted with a canceror any disorder involving, directly or indirectly, a cancer. Examples ofsubjects include mammals, e.g., humans, apes, monkeys, dogs, cows,horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenicnon-human animals. In an embodiment, the subject is a human, e.g., ahuman suffering from, at risk of suffering from, or potentially capableof suffering from cancers.

The terms “comprising” and “including” are used herein in theiropen-ended and non-limiting sense unless otherwise noted.

The terms “a” and “an” and “the” and similar references in the contextof describing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Where the plural form is used for compounds, salts, and the like, thisis taken to mean also a single compound, salt, or the like.

The terms “about” or “approximately” are generally understood by personsknowledgeable in the relevant subject area, but in certain circumstancescan mean within 20%, within 10%, or within 5% of a given value or range.Alternatively, especially in biological systems, the term “about” meanswithin about a log (i.e., an order of magnitude) or within a factor oftwo of a given value.

The term “synergistic effect” as used herein, refers to action of twoagents such as, for example, a compound of Formula I, or apharmaceutically acceptable salt thereof, and a CD38 inhibitor (e.g.,daratumumab), to produce an effect, for example, slowing the symptomaticprogression of cancer or symptoms thereof, which is greater than thesimple addition of the effects of each drug administered by itself. Asynergistic effect can be calculated, for example, using suitablemethods such as the Sigmoid-Emax equation (Holford, N. H. G. andScheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equationof Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. PatholPharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T.C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equationreferred to above can be applied to experimental data to generate acorresponding graph to aid in assessing the effects of the drugcombination. The corresponding graphs associated with the equationsreferred to above are the concentration-effect curve, isobologram curveand combination index curve, respectively. The pharmaceuticalcombinations provided herein, exhibit synergistic effects in connectionwith cell growth and viability in connection with myeloma and lymphomacell lines (see, e.g, Example 7).

The terms “combination,” “therapeutic combination,” or “pharmaceuticalcombination” as used herein refer to either a fixed combination in onedosage unit form, or non-fixed combination, or a kit of parts for thecombined administration where two or more therapeutic agents may beadministered independently, at the same time or separately within timeintervals, especially where these time intervals allow that thecombination partners show a cooperative, e.g., synergistic effect.

The term “combination therapy” refers to the administration of two ormore therapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single formulation having a fixedratio of active ingredients or in separate formulations (e.g., capsulesand/or intravenous formulations) for each active ingredient. Inaddition, such administration also encompasses use of each type oftherapeutic agent in a sequential or separate manner, either atapproximately the same time or at different times. Regardless of whetherthe active ingredients are administered as a single formulation or inseparate formulations, the drugs are administered to the same patient aspart of the same course of therapy. In any case, the treatment regimenwill provide beneficial effects in treating the conditions or disordersdescribed herein.

The term “histone deacetylase” or “HDAC” refers to enzymes that removethe acetyl groups from the lysine residues in core histones, thusleading to the formation of a condensed and transcriptionally silencedchromatin. There are currently 18 known histone deacetylases, which areclassified into four groups. Class I HDACs, which include HDAC1, HDAC2,HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs,which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are relatedto the yeast Hda1 gene. Class III HDACs, which are also known as thesirtuins are related to the Sir2 gene and include SIRT-1-7. Class IVHDACs, which contains only HDAC11, has features of both Class I and IIHDACs. The term “HDAC” refers to any one or more of the 18 known histonedeacetylases, unless otherwise specified.

The term “histone deacetylase inhibitor” (HDAC inhibitors, HDACi, HDIs)as used herein refers to a compound that selectively targets, decreases,or inhibits at least one activity of a histone deacetylase.

Histone Deacetylase Inhibitors

Provided herein are pharmaceutical combinations comprising a HDAC6inhibitor of Formula I (also referred to herein as “compounds of FormulaI”):

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R₁ is aryl or heteroaryl, each of which may be optionally substituted byOH, halo, or C₁₋₆-alkyl;

and

R is H or C₁₋₆-alkyl.

In an embodiment of the compound of Formula I, ring B is aryl. Invarious embodiments, R¹ is aryl or heteroaryl, each of which may beoptionally substituted by halo.

In an embodiment of Formula I, R₁ is an aryl that is substituted by OH,halo, or C₁₋₆-alkyl.

In another embodiment of Formula I, R₁ is C₅-C₇ aryl that is substitutedby OH, halo, or C₁₋₆-alkyl.

In another embodiment of Formula I, R₁ is C₄-C₇ heteroaryl that issubstituted by OH, halo, or C₁₋₆-alkyl.

In yet another embodiment of Formula I, R₁ is phenyl that is substitutedby OH, halo, or C₁₋₆-alkyl.

In yet another embodiment of Formula I, R₁ is phenyl that is substitutedby halo.

In yet another embodiment of Formula I, R₁ is phenyl that is substitutedby chloro.

In another embodiment of Formula I, ring B is C₅-C₇ aryl.

In another embodiment of Formula I, ring B is C₄-C₇ heteroaryl.

In yet another embodiment of Formula I, ring B is phenyl.

In a specific embodiment, the compound of Formula I is Compound A, or apharmaceutically acceptable salt thereof, or Compound B, or apharmaceutically acceptable salt thereof, as shown in Table 1:

TABLE 1

Compound A

Compound B

For convenience, the group of the HDAC6 inhibitors of Formula I and itssalts are collectively referred to as compounds of Formula I, meaningthat reference to compounds of Formula I will refer to any of thecompounds or pharmaceutically acceptable salt thereof in thealternative.

Compounds of Formula I (e.g., Compounds A and B) are known HDAC6inhibitors, and are described in PCT Pub. No. WO2011/091213, the contentof which is incorporated herein by reference in its entirety.

The preparation of Compounds A and B are also described herein asExample 1. Preferably, Compounds A and B are in the free base form.

CD38 Inhibitors

The term “CD38 inhibitor” as used herein refers to a compound thatselectively targets, decreases, or inhibits at least one activity ofCD38, a transmembrane glycoprotein. Non-limiting examples of CD38inhibitors include e.g., daratumumab (HuMax-CD38), isatuximab(SAR650984), and MOR202 (MOR03087), or pharmaceutically acceptable saltsthereof.

Disorders associated with dysregulated CD38 are, e.g., a variety oflymphoid tumors, notably multiple myeloma, AIDS-associated lymphomas,and post-transplant lympho-proliferations (Stevenson, G. T., Mol Med,12(11-12): 345-346, Nov.-Dec. 2006). In a particular embodiment, theCD38 inhibitor is daratumumab. Clinical trials investigating daratumumabin heavily-pretreated relapsed and/or refractory myeloma have beencompleted (Lonial, Sagar, et al., The Lancet, Vol. 387, No. 10027, p.1551-1560, 9 Apr. 2016). Further, daratumumab (also known as DARZALEX™)is approved by the FDA for the treatment of patients with multiplemyeloma who have received at least three prior lines of therapyincluding a proteasome inhibitor and an immunomodulatory agent, or whoare double-refractory to a PI and an immunomodulatory agent(http://www.accessdata.fda.gov/drugsatfda_docs/label/2015/761036s0001b1.pdf).

Any of the CD38 inhibitors can be used in the pharmaceuticalcombinations provided herein. In an exemplary embodiment, daratumumab isused. The amino acid sequences of daratumumab are shown in Table 2.

TABLE 2 Sequence of Selected Anti-CD38 Antibody Sequence ProteinIdentifier Sequence Daratumumab SEQ ID NO.: 1 EVQLLESGGGLVQPGGSLRLSCAVSGHeavy Chain FTFNSFAMSWVRQAPGKGLEWVSAIS GSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWF GEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK HCVRSEQ ID NO.: 2 EVQLLESGGGLVQPGGSLRLSCAVSG FTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSA HCDR1 SEQ ID NO.: 3 GFTFNSF HCDR2 SEQ ID NO.: 4SGSGGG HCDR3 SEQ ID NO.: 5 DKILWFGEPVFDY Daratumumab SEQ ID NO.: 6EIVLTQSPATLSLSPGERATLSCRASQ Light Chain SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFN RGEC LCVRSEQ ID NO.: 7 EIVLTQSPATLSLSPGERATLSCRASQ SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPPTFGQGTKVEI KR LCDR1SEQ ID NO.: 8 QSVSSYLA LCDR2 SEQ ID NO.: 9 DASNRAT LCDR3 SEQ ID NO.: 10QQRSNWPPT

Accordingly, in various embodiments of the pharmaceutical combination,the CD38 inhibitor is selected from the group consisting of daratumumab(HuMax-CD38), isatuximab (SAR650984), and MOR202 (MOR03087), orpharmaceutically acceptable salts thereof. In an embodiment of thepharmaceutical combination, the CD38 inhibitor is daratumumab. In anembodiment of the pharmaceutical combination, the CD38 inhibitor isisatuximab. In an embodiment of the pharmaceutical combination, the CD38inhibitor is MOR202.

In various embodiments of the pharmaceutical combination, the CD38inhibitor is a derivative, or a portion of any of the antibodiesdescribed herein (e.g., MOR202, isatuximab and the daratumumab anti-CD38antibody in Table 2). For example, the CD38 inhibitor is a derivative orfragment of MOR202 (MOR03087), isatuximab (SAR650984), and daratumumab(HuMax-CD38), or pharmaceutically acceptable salts thereof. In certainembodiments, the derivative or the fragment is a molecule sharing adistinct structure (e.g., 70%-99% similarity) and similar biologicalactivity in common with an antibody described herein. In certainembodiments, the derivative or the fragment comprises 70-75%, 75-80%,80-85%, 85-90%, 95-97%, or 97-99% sequence (i.e., nucleic acid and aminoacid) identity to a CD38 inhibitor described herein.

Methods of determining sequence identity are known in the art. Theidentity or homology may be the degree of identity between any givenquery sequence, for example, the percentage of nucleotide bases or aminoacid residues in the antibody sequence that are identical with theresidue of a corresponding sequence to which it is compared. Methods andcomputer programs for the alignment are available and well known in theart. See also international publication numbers WO2007084385 and WO2012113863.

The variable region typically refers to a portion of an antibody,generally, a portion of a light or heavy chain (e.g., about theamino-terminal 110 to 120 amino acids in a mature heavy chain and aboutthe amino-terminal 90 to 100 amino acids in a mature light chain) whichis used in the binding and specificity of a particular antibody for itsparticular antigen. The variability in sequence is concentrated in thoseregions called complementarity determining regions while the more highlyconserved regions in the variable domain are called framework regions(FR).

In certain embodiments the CD38 inhibitor of the invention comprises aheavy chain encoded by an amino acid sequence having at least 70%, 75%,80%, 85%, 90%, 95%, or 99% identity with the amino acid sequencerepresented by SEQ ID NO.: 1. The present invention also relates to aCD38 inhibitor comprising a light chain having an amino acid sequencewith at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with theamino acid sequence represented by SEQ ID NO.: 6. The present inventionalso relates to a CD38 inhibitor comprising a light chain having anamino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity with the amino acid sequence represented by SEQ ID NO.: 7. Invarious embodiments, the CD38 inhibitor comprises a heavy chain encodedby an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%,or 99% identity with the amino acid sequence represented by SEQ ID NO.:1; and comprises a light chain having an amino acid sequence with atleast 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acidsequence represented by SEQ ID NO.: 6.

In certain embodiments, the variable domains of heavy and light chainseach comprise complementarity determining regions (CDRs) which bind toCD38. See Kabat et al., 1987 Sequences of Proteins of ImmunologicalInterest, National Institute of Health, Bethesda, Md. For the heavychain variable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 106 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3. See for example U.S. Pat. No. 7,709,226 B2.

The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987) and (1991)) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as Kabat CDRs.Chothia and coworkers (Chothia and Lesk (1987) J. Mol. Biol.196:901-917) and Chothia et al. (1989) Nature 342:877-883) found thatcertain sub-portions within Kabat CDRs adopt nearly identical peptidebackbone conformations, despite having great diversity at the level ofamino acid sequence. These sub-portions were designated as L1, L2 and L3or H1, H2 and H3 where the “L” and the “H” designates the light chainand the heavy chains regions, respectively. These regions may bereferred to as Chothia CDRs, which have boundaries that overlap withKabat CDRs. Other boundaries defining CDRs overlapping with the KabatCDRs have been described by Padlan (1995) FASEB J. 9:133-139 andMacCallum (1996) J. Mol. Biol. 262(5):732-745. Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughparticular embodiments use Kabat or Chothia defined CDRs. See forexample international publication number WO2016149368.

In certain embodiments, the CD38 inhibitor includes the complementaritydetermining regions (CDR) sequences of an antibody described herein. Forexample, the CD38 inhibitor comprises a binding domain that comprisesone or more CDR regions found within an antibody described herein, e.g.,daratumumab and isatuximab. In certain embodiments, the CD38 inhibitorcomprises at least one CDR found within the heavy chain amino acidsequence of SEQ ID NO.: 1. In certain embodiments, the CD38 inhibitorcomprises one CDR, two CDRs or three CDRs found within the heavy chainamino acid sequence of SEQ ID NO.: 1. In certain embodiments, the CD38inhibitor comprises at least one CDR found within the heavy chain aminoacid sequence of SEQ ID NO.: 1. In certain embodiments, the CD38inhibitor comprises one CDR, two CDRs or three CDRs found within thelight chain amino acid sequence of SEQ ID NO.: 6. In various embodimentsthe CD38 inhibitor of the invention comprises at least one heavy chainCDR having at least 80%, 85%, 90%, 95%, or 99% identity with at leastone CDRs found within the amino acid sequence of SEQ ID NO.: 1. Invarious embodiments, the CD38 inhibitor comprises a light chain CDRhaving at least 80%, 85%, 90%, 95%, or 99% identity with at least oneCDR found within the amino acid sequence of SEQ ID NO.: 6.

Data provided herein shows that the combination therapy provided herein(e.g., HDAC6 inhibitors and a CD38 inhibitor) induces cell-mediatedcytotoxicity (see, e.g., Example 5). Further, the combination therapyprovided herein exhibits anti-tumor efficacy to a more significantextent relative to either single agent alone (see, e.g., Example 7).

Compounds of Formula I and the CD38 inhibitor, can be administered infree form or in pharmaceutically acceptable salt form.

Methods for Treating

Provided herein is a method for treating or preventing cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a pharmaceutical combinationprovided herein, i.e., a pharmaceutical combination comprising: (a) anHDAC6 inhibitor of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R₁ is aryl or heteroaryl, each of which may be optionally substituted byOH, halo, or C₁₋₆-alkyl;

and

R is H or C₁₋₆-alkyl; and

(b) a CD38 inhibitor.

In an embodiment, provided herein is a method for treating cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a pharmaceutical combinationprovided herein.

In a specific embodiment of the methods provided herein, the HDAC6inhibitor is Compound A, or a pharmaceutically acceptable salt thereof;and the CD38 inhibitor is daratumumab, or a pharmaceutically acceptablesalt thereof. In an embodiment of this embodiment, the method furthercomprises administration of pomalidomide. In an embodiment of thisembodiment, the method further comprises administration of pomalidomideand dexamethasone.

In another specific embodiment of the methods provided herein, the HDAC6inhibitor is Compound B, or a pharmaceutically acceptable salt thereof;and the CD38 inhibitor is daratumumab, or a pharmaceutically acceptablesalt thereof. In an embodiment of this embodiment, the method furthercomprises administration of pomalidomide. In an embodiment of thisembodiment, the method further comprises administration of pomalidomideand dexamethasone.

The method provided herein can be used for both solid tumors and liquidtumors. Further, depending on the tumor type and particular combinationused, a decrease of the tumor volume can be obtained. The combinationdisclosed herein is also suited to prevent the metastatic spread oftumors and the growth or development of micrometastases. The combinationdisclosed herein is suitable for the treatment of poor prognosispatients.

In various embodiments of the methods provided herein, the cancer is ahematologic cancer (e.g., lymphoma, leukemia, or myeloma). In furtherembodiments, the cancer is T-cell lymphoma or B-cell lymphoma.

In a further embodiment of any of the method provided herein, the canceris selected from multiple myeloma, amyloidosis, plasma cell myeloma,smoldering myeloma, mantle-cell lymphoma, diffuse large B-cell lymphoma,follicular lymphoma, chronic lymphocytic leukemia, non-Hodgkin'slymphoma, and Hodgkin's lymphoma.

In various embodiments of the method, the cancer is a non-solid tumor.In particular embodiments, the cancer is multiple myeloma.

In various embodiments, the cancer is resistant or refractory totreatment with at least one prior therapy. For example, when the canceris multiple myeloma, the myeloma can be resistant or refractory totreatment with thalidomide, pomalidomide, or lenalidomide, orpharmaceutically acceptable salts thereof. In other embodiments, thecancer is anti-CD38 naïve.

In some embodiments, provided herein is a method for treating a cancerin a subject in need thereof, comprising administering to the subject atherapeutically effective amount of Compound A, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of aCD38 inhibitor.

In another embodiment, provided herein is a method for treating ahematological cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anHDAC inhibitor of Formula I, or a pharmaceutically acceptable saltthereof, and a therapeutically effective amount of a CD38 inhibitor, ora pharmaceutically acceptable salt thereof. In some embodiments,provided herein is a method for treating a hematological cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of Compound A, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of aCD38 inhibitor, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for treating aleukemia in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of an HDAC inhibitor ofFormula I, or a pharmaceutically acceptable salt thereof, and atherapeutically effective amount of a CD38 inhibitor, or apharmaceutically acceptable salt thereof. In some embodiments, providedherein is a method for treating a leukemia in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of Compound A, or a pharmaceutically acceptable salt thereof, anda therapeutically effective amount of a CD38 inhibitor, or apharmaceutically acceptable salt thereof.

In another embodiment is a method for treating a cancer in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of Compound A, or a pharmaceutically acceptable saltthereof, and a therapeutically effective amount of daratumumab, or apharmaceutically acceptable salt thereof. This embodiment exhibitssynergy such that sub-therapeutic amounts of Compound A or ofdaratumumab can be used in the method.

In another embodiment is a method for treating a hematological cancer ina subject in need thereof, comprising administering to the subject atherapeutically effective amount of Compound A, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of adaratumumab, or a pharmaceutically acceptable salt thereof.

In another embodiment is a method for treating a leukemia in a subjectin need thereof, comprising administering to the subject atherapeutically effective amount of Compound A, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount ofdaratumumab, or a pharmaceutically acceptable salt thereof.

In yet another embodiment is a method for treating a cancer in a subjectin need thereof, comprising administering to the subject atherapeutically effective amount of Compound A, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of aCD38 inhibitor selected from the group consisting of daratumumab,isatuximab (SAR650984), and MOR202 (MOR03087), or pharmaceuticallyacceptable salts thereof.

In other embodiments, provided herein is a method for treating a cancerin a subject in need thereof, comprising administering to the subject atherapeutically effective amount of Compound B, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of aCD38 inhibitor, or a pharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to the subject atherapeutically effective amount of Compound B, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of aCD38 inhibitor selected from the group consisting of daratumumab,isatuximab (SAR650984), and MOR202 (MOR03087), or pharmaceuticallyacceptable salts thereof.

In some embodiments, provided herein is a method for treating ahematological cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount ofCompound B, or a pharmaceutically acceptable salt thereof, and atherapeutically effective amount of a CD38 inhibitor, or apharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for treating a leukemiain a subject in need thereof, comprising administering to the subject atherapeutically effective amount of Compound B, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of aCD38 inhibitor, or a pharmaceutically acceptable salt thereof.

In another embodiment is a method for treating a cancer in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of Compound B, or a pharmaceutically acceptable saltthereof, and a therapeutically effective amount of daratumumab, or apharmaceutically acceptable salt thereof. This embodiment exhibitssynergy such that sub-therapeutic amounts of Compound B or ofdaratumumab can be used in the method.

In another embodiment is a method for treating a hematological cancer ina subject in need thereof, comprising administering to the subject atherapeutically effective amount of Compound B, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount ofdaratumumab, or a pharmaceutically acceptable salt thereof.

In another embodiment is a method for treating a leukemia in a subjectin need thereof, comprising administering to the subject atherapeutically effective amount of Compound B, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount ofdaratumumab, or a pharmaceutically acceptable salt thereof.

For example, in one embodiment of the method, the HDAC inhibitor isadministered first, followed by the CD38 inhibitor. In anotherembodiment of the method, the CD38 inhibitor is administered first,followed by the HDAC inhibitor.

In various embodiments of the methods, the method further comprisesadministering to the subject a therapeutically effective amount ofdexamethasone.

The subject considered herein is typically a human. However, the subjectcan be any mammal for which treatment is desired. Thus, the methodsdescribed herein can be applied to both human and veterinaryapplications.

In various embodiments of the methods, the method further comprisesadministering to the subject a therapeutically effective amount of animmunomodulatory drug. In an embodiment, the immunomodulatory drug ispomalidomide. In another embodiment, the immunomodulatory drug islenalidomide.

Pharmaceutical Combinations and Compositions

Provided herein is a pharmaceutical combination comprising a histonedeacetylase inhibitor 6 (HDAC6) inhibitor and a CD38 inhibitor.

In an aspect, provided herein is a pharmaceutical combinationcomprising:

(a) an HDAC6 inhibitor of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl

R₁ is aryl or heteroaryl, each of which may be optionally substituted byOH, halo, or C₁₋₆-alkyl; and

(b) a CD38 inhibitor, or a pharmaceutically acceptable salt thereof.

In various embodiments of the pharmaceutical combination, ring B isaryl.

In various embodiments of the pharmaceutical combination, R¹ is aryl orheteroaryl, each of which may be optionally substituted by halo.

In various embodiments of the pharmaceutical combination, the compoundof Formula I is Compound A, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound of Formula I is Compound B,or a pharmaceutically acceptable salt thereof.

In various embodiments of the pharmaceutical combination, thecombination further comprises a compound selected from thalidomide or ananalog thereof, or a pharmaceutically acceptable salt thereof. In otherembodiments, the compound is selected from thalidomide, pomalidomide,and lenalidomide, or a pharmaceutically acceptable salt thereof. Inexemplary embodiments, the compound is pomalidomide.

In various embodiments, the CD38 inhibitor is an inhibitory antibody. Inan embodiment, the CD38 inhibitor comprises an amino acid sequenceselected from the group consisting of SEQ ID NO.:1 and SEQ ID NO.: 6. Inan embodiment, the CD38 inhibitor is selected from the group consistingof daratumumab, isatuximab (SAR650984), and MOR202 (MOR03087), orpharmaceutically acceptable salts thereof. In a particular embodiment,the CD38 inhibitor is daratumumab, or a bioequivalent, or apharmaceutically acceptable salt thereof.

In various embodiments of the pharmaceutical combination, the HDAC6inhibitor and the CD38 inhibitor are in the same formulation.Alternatively, the HDAC6 inhibitor and the CD38 inhibitor are inseparate formulations.

In various embodiments, the pharmaceutical combination is for use intreating cancer in a subject in need thereof.

In an embodiment, the combination of the invention is used for thetreatment of cancer comprising administering to the subject acombination therapy, comprising an effective amount of the HDAC6inhibitor (i.e., compounds of Formula I) and an effective amount of aCD38 inhibitor. Preferably, these compounds are administered attherapeutically effective dosages which, when combined, provide abeneficial effect. The administration can comprise the separateadministration of each component, either simultaneously, orsequentially.

In various embodiments, the pharmaceutical combination is for use in thepreparation of a medicament for the treatment of cancer.

The pharmaceutical combination provided herein can also inhibit thegrowth of both solid tumors and liquid tumors. Further, depending on thetumor type and particular combination used, a decrease of the tumorvolume can be obtained. The combination disclosed herein is also suitedto prevent the metastatic spread of tumors and the growth or developmentof micrometastases. The combination disclosed herein is suitable for thetreatment of poor prognosis patients.

In various embodiments, the cancer is a hematologic cancer (e.g., alymphoma, leukemia, or myeloma). In further embodiments, the cancer is aT-cell lymphoma or a B-cell lymphoma.

In an embodiment of any of the pharmaceutical combinations providedherein, the cancer is selected from multiple myeloma, amyloidosis,plasma cell myeloma, smoldering myeloma, mantle-cell lymphoma, diffuselarge B-cell lymphoma, follicular lymphoma, chronic lymphocyticleukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

In various embodiments of the pharmaceutical combination, the cancer isa non-solid cancer. In particular embodiments, the cancer is multiplemyeloma.

In various embodiments, the cancer is resistant or refractory totreatment with at least one prior therapy. In other embodiments, thecancer or subject is anti-CD38 naïve.

In various embodiments of the pharmaceutical combination, thecombination further comprises a therapeutically effective amount of animmunomodulatory drug. In an embodiment, the immunomodulatory drug ispomalidomide. In another embodiment, the immunomodulatory drug islenalidomide.

Also provided herein are pharmaceutical compositions comprising ahistone deacetylase inhibitor (HDAC) inhibitor and a CD38 inhibitor.

As used herein, term “pharmaceutical composition” is defined herein torefer to a mixture or solution containing the therapeutic agent(s) to beadministered to a subject, e.g., a mammal or human, in order to preventor treat a particular disease or condition affecting the mammal.

In an aspect, provided herein is a pharmaceutical composition comprising

(a) an HDAC6 inhibitor of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R₁ is aryl or heteroaryl, each of which may be optionally substituted byOH, halo, or C₁₋₆-alkyl;

and

R is H or C₁₋₆-alkyl; and

(b) a CD38 inhibitor.

In an embodiment of the composition, ring B is aryl. In variousembodiments, R₁ is aryl or heteroaryl, each of which is substituted byhalo.

In another embodiment of the composition, the HDAC6 inhibitor isCompound A, or a pharmaceutically acceptable salt thereof. In anotherembodiment of the composition, the HDAC6 inhibitor is Compound B, or apharmaceutically acceptable salt thereof.

In various embodiments of the pharmaceutical composition, the CD38inhibitor is an inhibitory antibody, e.g., daratumumab (HuMax-CD38),isatuximab (SAR650984), and MOR202 (MOR03087), or pharmaceuticallyacceptable salts thereof. In particular embodiments, the CD38 inhibitoris daratumumab, or a pharmaceutically acceptable salt thereof.

In an embodiment of the composition, the pharmaceutical compositionfurther comprises one or more excipients. As used herein, the term“pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

The pharmaceutical composition may contain, from about 0.1% to about99.9%, preferably from about 1% to about 60%, of the therapeuticagent(s).

Suitable pharmaceutical compositions for the combination therapy forenteral or parenteral administration are, for example, those in unitdosage forms, such as sugar-coated tablets, tablets, capsules orsuppositories, or ampoules. If not indicated otherwise, these areprepared in a manner known per se, for example by means of variousconventional mixing, comminution, direct compression, granulating,sugar-coating, dissolving, lyophilizing processes, melt granulation, orfabrication techniques readily apparent to those skilled in the art. Itwill be appreciated that the unit content of a combination partnercontained in an individual dose of each dosage form need not in itselfconstitute an effective amount since the necessary effective amount maybe reached by administration of a plurality of dosage units.

Administration/Dose

The method of treating cancer according to the disclosure providedherein can comprise (i) administration of the HDAC6 inhibitor (a) infree or pharmaceutically acceptable salt form and (ii) administration ofa CD38 inhibitor (b) in free or pharmaceutically acceptable salt formsimultaneously or sequentially, in any order, in jointly therapeuticallyeffective amounts, preferably in synergistically effective amounts,e.g., in daily or intermittent dosages. The individual combinationpartners of the pharmaceutical combination provided herein can beadministered separately at different times during the course of therapyor concurrently in divided or single combination forms. The methodprovided herein is therefore to be understood as embracing all suchregimens of simultaneous or alternating treatment and the term“administering” is to be interpreted accordingly. Compounds of Formula Ican be orally administered in an amount from about 10 mg to about 1000mg (including e.g., about 10 mg to about 500 mg) per day in single ormultiple doses. Thus, in an embodiment of the methods of treatmentprovided herein, the compound of Formula I is administered at a dosageof about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg,100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg,190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg,280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg,370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg,460 mg, 470 mg, 480 mg, 490 mg, or 500 mg per day. In a furtherembodiment, the compound of Formula I is administered at a dosage of 20mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120mg, 130 mg, 140 mg, 150 mg, 160 mg, 180 mg, or 200 mg per day.

In an embodiment of the pharmaceutical combination, Compound A is in anamount from 600 mg to 3000 mg (e.g., about 600, about 800, about 1000,about 1200, about 1400, about 1600, about 1800, about 2000 mg). In afurther embodiment of the pharmaceutical combination, Compound A is inan amount from 600 mg to 2000 mg. In a preferred embodiment of thepharmaceutical combination, Compound A is in an amount from 180 mg to480 mg.

In another embodiment of the pharmaceutical combination, Compound A isin an amount from 5 mg to 600 mg (e.g., about 5, about 25, about 50,about 100, about 200, about 300, about 400, about 500, about 600 mg). Inyet another embodiment of the pharmaceutical combination Compound A is10 mg to 200 mg.

In an embodiment of the pharmaceutical combination, Compound B is in anamount from 600 mg to 3000 mg (e.g., about 600, about 800, about 1000,about 1200, about 1400, about 1600, about 1800, about 2000 mg). In afurther embodiment of the pharmaceutical combination, Compound B is inan amount from 600 mg to 2000 mg. In a preferred embodiment of thepharmaceutical combination, Compound B is in an amount from 180 mg to480 mg.

In another embodiment of the pharmaceutical combination, Compound B isin an amount from 5 mg to 600 mg (e.g., about 5, about 25, about 50,about 100, about 200, about 300, about 400, about 500, about 600 mg). Inyet another embodiment of the pharmaceutical combination Compound B is10 mg to 200 mg.

In an embodiment of the pharmaceutical combination, the CD38 inhibitoris in an amount from 600 mg to 3000 mg (e.g., about 600, about 800,about 1000, about 1200, about 1400, about 1600, about 1800, about 2000mg). In a further embodiment of the pharmaceutical combination, the CD38inhibitor is in an amount from 600 mg to 2000 mg.

In another embodiment of the pharmaceutical combination, daratumumab isin an amount from 5 mg to 600 mg (e.g., about 5, about 25, about 50,about 100, about 200, about 300, about 400, about 500, about 600 mg). Inyet another embodiment of the pharmaceutical combination, daratumumab is10 mg to 200 mg. In an embodiment of the pharmaceutical combination,daratumumab is in an amount from 600 mg to 3000 mg (e.g., about 600,about 800, about 1000, about 1200, about 1400, about 1600, about 1800,about 2000 mg). In a further embodiment of the pharmaceuticalcombination, daratumumab is in an amount from 600 mg to 2000 mg.

In another embodiment of the pharmaceutical combination, daratumumab isin an amount from 5 mg to 600 mg (e.g., about 5, about 25, about 50,about 100, about 200, about 300, about 400, about 500, about 600 mg). Inyet another embodiment of the pharmaceutical combination, daratumumab is10 mg to 200 mg.

In another embodiment of the pharmaceutical combination, daratumumab isin an amount from 5 mg/kg to 20 mg/kg (e.g., about 5 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg).

The effective dosage of each of the combination partners employed in thecombination provided herein may vary depending on the particularcompound or pharmaceutical composition employed, the mode ofadministration, the condition being treated, and the severity of thecondition being treated. Thus, the dosage regimen of the combination ofthe invention is selected in accordance with a variety of factorsincluding the route of administration and the renal and hepatic functionof the patient.

The optimum ratios, individual and combined dosages, and concentrationsof the combination partners (e.g., compound of Formula I and a CD38inhibitor) of the combination provided herein that yield efficacywithout toxicity are based on the kinetics of the therapeutic agents'availability to target sites, and are determined using methods known tothose of skill in the art.

In an embodiment of the pharmaceutical combination, the ratio of thecompound of Formula I to the CD38 inhibitor is in the range of700:1-1:40. In another embodiment, the ratio of the compound of FormulaI to the CD38 inhibitor is in the range of 2:1 to 1:2, for example, 2:1,1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to1:1, for example, 3:1, 2:1 or 1:1; 4:1 to 1:1, for example, 4:1, 3:1,2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.

In another embodiment of the pharmaceutical combination, the ratio ofCompound A to the CD38 inhibitor is in the range of 700:1-1:40. Inanother embodiment, the ratio of Compound A to the CD38 inhibitor is inthe range of 2:1 to 1:2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1,for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or1:1; 4:1 to 1:1, for example, 4:1, 3:1, 2:1 or 1:1; or 30:1 to 10:1, forexample, 30:1, 20:1 or 10:1.

In another embodiment of the pharmaceutical combination, the ratio ofCompound B to the CD38 inhibitor is in the range of 700:1-1:40. Inanother embodiment, the ratio of Compound B to the CD38 inhibitor is inthe range of 2:1 to 1:2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1,for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.

In another embodiment of the pharmaceutical combination, the ratio ofCompound A to the daratumumab is in the range of 700:1-1:40. In anotherembodiment, the ratio of Compound A to the daratumumab is in the rangeof 2:1 to 1:2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, forexample, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.

In another embodiment of the pharmaceutical combination, the ratio ofCompound B to the daratumumab is in the range of 700:1-1:40. In anotherembodiment, the ratio of Compound B to the daratumumab is in the rangeof 2:1 to 1:2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, forexample, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.

In an embodiment, the pharmaceutical combination or composition, orboth, provided herein display a synergistic effect. In anotherembodiment, the pharmaceutical combination or composition isadministered at dosages that would not be effective when one or both ofthe HDAC inhibitor and the CD38 inhibitor is administered alone, butwhich amounts are effective in combination.

In determining a synergistic interaction between one or more components,the optimum range for the effect and absolute dose ranges of eachcomponent for the effect may be definitively measured by administrationof the components over different w/w ratio ranges and doses to patientsin need of treatment. For humans, the complexity and cost of carryingout clinical studies on patients may render impractical the use of thisform of testing as a primary model for synergy. However, the observationof synergy in certain experiments can be predictive of the effect inother species, and animal models exist may be used to further quantify asynergistic effect. The results of such studies can also be used topredict effective dose ratio ranges and the absolute doses and plasmaconcentrations.

In a further embodiment, provided herein is a synergistic combinationfor administration to a subject comprising the combination of theinvention, where the dose range of each component corresponds to thesynergistic ranges suggested in a suitable tumor model or clinicalstudy.

The effective dosage of each of the combination partners may requiremore frequent administration of one of the compound(s) as compared tothe other compound(s) in the combination. Therefore, to permitappropriate dosing, packaged pharmaceutical products may contain one ormore dosage forms that contain the combination of compounds, and one ormore dosage forms that contain one of the combinations of compounds, butnot the other compound(s) of the combination.

When the combination partners, which are employed in the combination ofthe invention, are applied in the form as marketed as single drugs,their dosage and mode of administration can be in accordance with theinformation provided on the package insert of the respective marketeddrug, if not mentioned herein otherwise.

The optimal dosage of each combination partner for treatment of a cancercan be determined empirically for each individual using known methodsand will depend upon a variety of factors, including, though not limitedto: the degree of advancement of the disease; the age, body weight,general health, gender and diet of the individual; the time and route ofadministration; and other medications the individual is taking. Optimaldosages may be established using routine testing and procedures that arewell known in the art.

The amount of each combination partner that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the individual treated and the particular mode of administration.In some embodiments the unit dosage forms containing the combination ofagents as described herein will contain the amounts of each agent of thecombination that are typically administered when the agents areadministered alone.

Frequency of dosage may vary depending on the compound used and theparticular condition to be treated or prevented. Patients may generallybe monitored for therapeutic effectiveness using assays suitable for thecondition being treated or prevented, which will be familiar to those ofordinary skill in the art.

Also provided herein is a commercial package comprising, as therapeuticagents, the pharmaceutical combination provided herein, together withinstructions for simultaneous, separate or sequential administrationthereof for use in the delay of progression or treatment of a cancer.

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

EXAMPLES

The following Examples illustrate the invention described above; theyare not, however, intended to limit the scope of the invention in anyway. The beneficial effects of the pharmaceutical combination of thepresent invention can also be determined by other test models known assuch to the person skilled in the pertinent art.

Example 1: Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound A) and2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound B) I. Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound A)

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol),compound 1 (7.5 g, 40 mmol), and K₂CO₃ (11 g, 80 mmol) in DMF (100 ml)was degassed and stirred at 120° C. under N₂ overnight. The reactionmixture was cooled to r.t. and diluted with EtOAc (200 ml), then washedwith saturated brine (200 ml×3). The organic layers were separated anddried over Na₂SO₄, evaporated to dryness and purified by silica gelchromatography (petroleum ethers/EtOAc—10/1) to give the desired productas a white solid (6.2 g, 64%).

Synthesis of Intermediate 3: A mixture of compound 2 (6.2 g, 25 mmol),iodobenzene (6.12 g, 30 mmol), Cul (955 mg, 5.0 mmol), Cs₂CO₃ (16.3 g,50 mmol) in TEOS (200 ml) was degassed and purged with nitrogen. Theresulting mixture was stirred at 140° C. for 14 hrs. After cooling tor.t., the residue was diluted with EtOAc (200 ml). 95% EtOH (200 ml) andNH₄F—H₂O on silica gel [50 g, pre-prepared by the addition of NH₄F (100g) in water (1500 ml) to silica gel (500 g, 100-200 mesh)] was added,and the resulting mixture was kept at r.t. for 2 hrs. The solidifiedmaterials were filtered and washed with EtOAc. The filtrate wasevaporated to dryness and the residue was purified by silica gelchrornatography (petroleum ethers/EtOAc=10/1) to give a yellow solid (3g, 38%).

Synthesis of Intermediate 4:2N NaOH (200 ml) was added to a solution ofcompound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirredat 60° C. for 30 min. After evaporation of the solvent, the solution wasneutralized with 2N HCl to give a white precipitate. The suspension wasextracted with EtOAc (2×200 ml), and the organic layers were separated,washed with water (2×100 ml), brine (2×100 ml), and dried over Na₂SO₄.Removal of the solvent gave a brown solid (2.5 g, 92%).

Synthesis of Intermediate 6: A mixture of compound 4 (2.5 g, 8.58 mmol),compound 5 (2.52 g, 12.87 mmol), HATU (3.91 g, 10.30 mmol), and DIPEA(4.43 g, 34.32 mmol) was stirred at r.t. overnight. After the reactionmixture was filtered, the filtrate was evaporated to dryness and theresidue was purified by silica gel chromatography (petroleumethers/EtOAc=2/1) to give a brown solid (2 g, 54%).

Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide:A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0° C. for 10 min.Hydroxylamine (50%) (10 ml) was cooled to 0° C. and added to themixture. The resulting mixture was stirred at r.t. for 20 min. Afterremoval of the solvent, the mixture was neutralized with 1M HCl to givea white precipitate. The crude product was filtered and purified bypre-HPLC to give a white solid (950 mg, 48%).

II. Synthetic Route 1:2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound B)

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol),ethyl 2-chloropyrimidine-5-carboxylate 1 (7.5 g, 40 mmol), K₂CO₃ (11 g,80 mmol) in DMF (100 ml) was degassed and stirred at 120° C. under N₂overnight. The reaction mixture was cooled to rt and diluted with EtOAc(200 ml), then washed with saturated brine (200 ml×3). The organic layerwas separated and dried over Na₂SO₄, evaporated to dryness and purifiedby silica gel chromatography (petroleum ethers/EtOAc=10/1) to give thedesired product as a white solid (6.2 g, 64%).

Synthesis of Intermediate 3: A mixture of compound 2 (69.2 g, 1 equiv.),1-chloro-2-iodobenzene (135.7 g, 2 equiv.), Li₂CO₃ (42.04 g, 2 equiv.),K₂CO₃ (39.32 g, 1 equiv.), Cu (1 equiv. 45 μm) in DMSO (690 ml) wasdegassed and purged with nitrogen. The resulting mixture was stirred at140° C. for 36 hours. Work-up of the reaction gave compound 3 at 93%yield.

Synthesis of Intermediate 4: 2N NaOH (200 ml) was added to a solution ofthe compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture wasstirred at 60° C. for 30 min. After evaporation of the solvent, thesolution was neutralized with 2N HCl to give a white precipitate. Thesuspension was extracted with EtOAc (2×200 ml), and the organic layerwas separated, washed with water (2×100 ml), brine (2×100 ml), and driedover Na₂SO₄. Removal of solvent gave a brown solid (2.5 g, 92%).

Synthesis of Intermediate 5: A procedure analogous to the Synthesis ofIntermediate 6 in Part I of this Example was used.

Synthesis of2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide:A procedure analogous to the Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamidein Part I of this Example was used.

III. Synthetic Route 2:2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound B)

Step (1): Synthesis of Compound 11: Ethyl2-chloropyrimidine-5-carboxylate (7.0 Kgs), ethanol (60 Kgs),2-Chloroaniline (9.5 Kgs, 2 eq) and acetic acid (3.7 Kgs, 1.6 eq) werecharged to a reactor under inert atmosphere. The mixture was heated toreflux. After at least 5 hours the reaction was sampled for HPLCanalysis. When analysis indicated reaction completion, the mixture wascooled to 70±5° C. and N,N-Diisopropylethylamine (DIPEA) was added. Thereaction was then cooled to 20±5° C. and the mixture was stirred for anadditional 2-6 hours. The resulting precipitate is filtered and washedwith ethanol (2×6 Kgs) and heptane (24 Kgs). The cake is dried underreduced pressure at 50±5° C. to a constant weight to produce 8.4 Kgscompound 11 (81% yield and 99.9% purity.

Step (2): Synthesis of Compound 3: Copper powder (0.68 Kgs, 1 eq, <75micron), potassium carbonate (4.3 Kgs, 1.7 eq), and dimethyl sulfoxide(DMSO, 12.3 Kgs) were added to a reactor (vessel A). The resultingsolution was heated to 120±5° C. In a separate reactor (vessel B), asolution of compound 11 (2.9 Kgs) and iodobenzene (4.3 Kgs, 2 eq) inDMSO (5.6 Kgs) was heated at 40±5° C. The mixture was then transferredto vessel A over 2-3 hours. The reaction mixture was heated at 120±5° C.for 8-24 hours, until HPLC analysis determined that ≤1% compound 11 wasremaining.

Step (3): Synthesis of Compound 4: The mixture of Step (2) was cooled to90-100° C. and purified water (59 Kgs) was added. The reaction mixturewas stirred at 90-100° C. for 2-8 hours until HPLC showed that 51%compound 3 was remaining. The reactor was cooled to 25° C. The reactionmixture was filtered through Celite, then a 0.2 micron filter, and thefiltrate was collected. The filtrate was extracted with methyl t-butylether twice (2×12.8 Kgs). The aqueous layer was cooled to 0-5° C., thenacidified with 6N hydrochloric acid (HCl) to pH 2-3 while keeping thetemperature<25° C. The reaction was then cooled to 5-15° C. Theprecipitate was filtered and washed with cold water. The cake was driedat 45-55° C. under reduced pressure to constant weight to obtain 2.2 kg(65% yield) compound 4 in 90.3% AUC purity.

Step (4): Synthesis of Compound 5: Dichloromethane (40.3 Kgs), DMF (33g, 0.04 eq) and compound 4 (2.3 Kg) were charged to a reaction flask.The solution was filtered through a 0.2 μm filter and was returned tothe flask. Oxalyl chloride (0.9 Kgs, 1 eq) was added via addition funnelover 30-120 minutes at <30° C. The batch was then stirred at <30° C.until reaction completion (compound 4≤3%) was confirmed by HPLC. Next,the dichloromethane solution was concentrated and residual oxalylchloride was removed under reduced pressure at <40° C. When HPLCanalysis indicated that <0.10% oxalyl chloride was remaining, theconcentrate was dissolved in fresh dichloromethane (24 Kgs) andtransferred back to the reaction vessel (Vessel A).

A second vessel (Vessel B) was charged with Methyl 7-aminoheptanoatehydrochloride (Compound A1, 1.5 Kgs, 1.09 eq), DIPEA (2.5 Kgs, 2.7 eq),4 (Dimethylamino)pyridine (DMAP, 42 g, 0.05 eq), and DCM (47.6 Kgs). Themixture was cooled to 0-10° C. and the acid chloride solution in VesselA was transferred to Vessel B while maintaining the temperature at 5° C.to 10° C. The reaction is stirred at 5-10° C. for 3 to 24 hours at whichpoint HPLC analysis indicated reaction completion (compound 4≤55%). Themixture was then extracted with a 1M HCl solution (20 Kgs), purifiedwater (20 Kgs), 7% sodium bicarbonate (20 Kgs), purified water (20 Kgs),and 25% sodium chloride solution (20 Kgs). The dichloromethane was thenvacuumdistilled at <40° C. and chased repeatedly with isopropyl alcohol.When analysis indicated that <1 mol % DCM was remaining, the mixture wasgradually cooled to 0-5° C. and was stirred at 0-5° C. for an at least 2hours. The resulting precipitate was collected by filtration and washedwith cold isopropyl alcohol (6.4 Kgs). The cake was sucked dry on thefilter for 4-24 hours, then was further dried at 45-55° C. under reducedpressure to constant weight. 2.2 Kgs (77% yield) was isolated in 95.9%AUC purity method and 99.9 wt %.

Step (5): Synthesis of Compound (I): Hydroxylamine hydrochloride (3.3Kgs, 10 eq) and methanol (9.6 Kgs) were charged to a reactor. Theresulting solution was cooled to 0-5° C. and 25% sodium methoxide (11.2Kgs, 11 eq) was charged slowly, maintaining the temperature at 0-10° C.Once the addition was complete, the reaction was mixed at 20° C. for 1-3hours and filtered, and the filter cake was washed with methanol (2×2.1Kgs). The filtrate (hydroxylamine free base) was returned to the reactorand cooled to 0±5° C. Compound 5 (2.2 Kgs) was added. The reaction wasstirred until the reaction was complete (compound 5≤2%). The mixture wasfiltered and water (28 Kgs) and ethyl acetate (8.9 Kgs) were added tothe filtrate. The pH was adjusted to 8-9 using 6N HCl then stirred forup to 3 hours before filtering. The filter cake was washed with coldwater (25.7 Kgs), then dried under reduced pressure to constant weight.The crude solid compound (I) was determined to be Form IV/Pattern D.

The crude solid (1.87 Kgs) was suspended in isopropyl alcohol (IPA, 27.1Kg). The slurry was heated to 75±5° C. to dissolve the solids. Thesolution was seeded with crystals of Compound (1) (Form I/Pattern A),and was allowed to cool to ambient temperature. The resultingprecipitate was stirred for 1-2 hours before filtering. The filter cakewas rinsed with IPA (2×9.5 Kgs), then dried at 45-55° C. to constantweight under reduced pressure to result in 1.86 kg crystalline whitesolid Compound (I) in 85% yield and 99.5% purity (AUC %, e.g., by theHPLC method of Table 3).

TABLE 3 HPLC Method Column Zorbax Eclipse XDB-C18, 4.6 mm × 150 mm, 3.5μm Column 40° C. Temperature UV Detection Bandwidth 4 nm, Reference off,272 nm Wavelength Flow rate 1.0 mL/min Injection 10 μL with needle washVolume Mobile 0.05% trifluoroacetic acid (TFA) in purified water Phase AMobile 0.04% TFA in acetonitrile Phase B Data 40.0 min Collection RunTime 46.0 min Time (min) Mobile Phase A Mobile Phase B Gradient 0.0 98% 2% 36.0  0% 100% 40.0  0% 100% 40.1 98%  2% 46.0 98%  2%

Example 2: HDAC Enzyme Assay

Compound B was tested first by diluting the compound in DMSO to 50 foldthe final concentration and a ten point three fold dilution series wasmade. The compound was diluted in assay buffer (50 mM HEPES, pH 7.4, 100mM KCl, 0.001% Tween-20, 0.05% BSA, 20 μM TCEP) to 6 fold their finalconcentration. The HDAC enzymes (purchased from BPS Biosciences; SanDiego, Calif.) were diluted to 1.5 fold their final concentration inassay buffer. The tripeptide substrate and trypsin at 0.05 μM finalconcentration were diluted in assay buffer at 6 fold their finalconcentration. The final enzyme concentrations used in these assays were3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml(HDAC6). The final substrate concentrations used were 16 μM (HDAC1), 10μM (HDAC2), 17 μM (HDAC3) and 14 μM (HDAC6). Five μl of compound and 20μl of enzyme were added to wells of a black, opaque 384 well plate induplicate. Enzyme and compound were incubated together at roomtemperature for 10 minutes. Five 41 of substrate was added to each well,the plate was shaken for 60 seconds and placed into a Victor 2microtiter plate reader. The development of fluorescence was monitoredfor 60 min and the linear rate of the reaction was calculated. The IC₅₀was determined using Graph Pad Prism by a four parameter curve fit. SeeTable 1 for IC₅₀ associated with Compounds A and B.

Example 3: Methods Cell Lines

MM.1S, NCI-H929, RPM18226 and U266 cell lines were obtained fromAmerican Type Culture Collection (Manassas, Va.). All cell lines werecultured in RPMI-1640 containing 10% fetal bovine serum (FBS, SigmaChemical Co., St. Louis, Mo.), 2 μM L-glutamine, 100 U/ml penicillin,and 100 μg/ml streptomycin (GIBCO, Grand Island, N.Y.).

Reagents

Compound A and Compound B were provided by Acetylon Pharmaceuticals.Anti-CD38 antibodies were provided by Acetylon Pharmaceuticals. For invivo tumor xenograft studies, daratumumab was provided by Charles RiverLaboratories.

Antibody-Dependent Cell-Mediated (ADCC) Assay Using MM Cell Lines InVitro

Multiple myeloma cells were incubated with Calcein-AM (Invitrogen, 2Ng/ml/1 million cells) for 30 min at 37° C. After washing, the cellswere used for target cells. Mononuclear cells were obtained from healthyvolunteer peripheral blood by Ficoll-Paque density centrifugation andused for effector cells. Both target and effector cells (E/T=15-20) wereincubated for 3-4 h and culture supernatant were subjected tomeasurement of fluorescence using a 490 nm excitation filter and a 520emission filter. Percent specific lysis was calculated as follows,

% specific lysis=[experiment fluorescence−spontaneous fluorescence (noeffector)]/[complete lysis (100% killing by detergent)−spontaneousfluorescence (no effector)]×100

Flow Cytometry-Based Ex Vivo ADCC Assay

After bone marrow aspiration, fresh bone marrow mononuclear cells (MNC)were obtained by ammonium chloride based-lyses of red cells followed bywashing in phosphate-buffered saline (PBS). For ADCC assays, cells wereimmediately incubated with anti-CD38 antibody (daratumumab, 1 μg/mL) inthe presence of Compound A or Compound B in complete medium in 96-wellplate. The cells were incubated for 72 h at 37° C. in 5% CO2. Theviability of primary MM cells in BM MNC was determined by near infrared(n-IR) viability dyes. The surviving CD138+MM cells were enumerated inthe presence of Flow-Count Fluorospheres (Beckman Coulter, CA, USA), todetermine absolute numbers of viable MM cells. The percentage ofdaratumumab-mediated ADCC was then calculated using the followingformula: % lysis cells=100−[(absolute number of surviving CD138+ cellsin the presence of daratumumab/absolute number of surviving CD138+ cellsin the presence of control antibody)×100%]. Phenotypic analyses of MNCwere performed using a single 5-color combination of monoclonalantibodies containing CD138-PE/CD38-APC/n-IR-viability (BioLegend, SanDiego, Calif., USA). Plasma cells in BM MNC were identified by CD138-PEand CD38-APC staining, and expression of CD38 on plasma cells weredetermined (BD Biosciences, San Jose, Calif., USA). After incubation,cells were washed twice in PBS containing 2% BSA, and then resuspendedin PBS for analysis on a Fortessa cytometer (Becton Dickinson, MountainView, Calif.). MM cell lines were also applied to this method in someexperiments.

Example 4: Compound a and Compound B Increase CD38 Surface Level inMultiple Myeloma

To determine the effect of Compound A and Compound B on the surfacelevel of CD38 in multiple myeloma cells, RPMI-8226 (FIG. 1A) and MM.1S(FIG. 11B) cells were treated with Compound A and Compound B at variousdoses for 48 hours. Surface levels of CD38 were determined by FACSanalysis. Data show that both Compound A and Compound B increased thepercentage of CD38 positive cells.

Example 5: Compound a Enhances Anti-CD38-Induced Antibody-DependentCell-Mediated Cytotoxicity

To determine the effect of Compound A on antibody-dependentcell-mediated cytotoxicity in H929 cells, cells were incubated with 0.5μM-2 μM Compound A for 24 hours. Cells were stained with Calcein-AM andincubated with peripheral blood mononuclear cells at a ratio of 20:1 for3 hours. Percent specific lysis (FIG. 2A) and percent augmentation (FIG.2B) increased as concentration of Compound A increased. Similarly, H929cells were incubated with pomalidomide at 0.1 μM or 1 μM and/or CompoundA at 0.25 μM, 0.5 μM, or 1 μM for 48 hours followed by subsequentincubation with effector peripheral blood mononuclear cells with orwithout anti-CD38 antibody for 3 hours. The greatest anti-CD38antibody-dependent cell-mediated toxicity was observed in cellsincubated with the combination of pomalidomide and Compound A (FIG. 5).

Example 6: Compound B Enhances Anti-CD38 Ab Induced-ADCC in AutologousSetting

To determine the effect of Compound B on ADCC of mononuclear cells in anautologous setting, cells from multiple myeloma patients' bone marrowaspirates were cultured with anti-CD38 antibody (0.5 μg/ml) with orwithout Compound B (0.5 μM) for 72 hours. It was observed that theanti-CD38 antibody dependent cell mediated cytotoxicity increased insamples treated with Compound B (FIG. 3A). The average cytotoxicity(mean±SD) was calculated from 6 patients' samples (see also FIG. 3B).

Example 7: Compound B Enhances Anti-Tumor Efficacy in Combination withDaratumumab

To determine the anti-tumor efficacy of the combination of Compound Band the anti-CD38 antibody daratumumab were used in a mouse model oflymphoma. Daudi Burkitt's lymphoma cells were implanted subcutaneouslyin CB17 SCID mice and allowed to form tumors. Mice were then randomizedto treatment with vehicle, Compound B in combination with non-targetingIgG control antibody; daratumumab alone; or Compound B in combinationwith daratumumab. Combination treatment with Compound B and daratumumabresulted in greater tumor growth suppression relative to either singleagent (FIG. 4A). Additionally, combination treatment resulted in agreater partial and complete response as measured by tumor volume (FIG.4B). The tumor volume of individual animals showed a high rate of tumorregression even after completion of dosing (FIG. 4C).

Example 8: Compound B Enhances Antibody-Dependent Cell-MediatedCytotoxicity

To assess the effect of Compound B in combination with pomalidomide onADCC using anti-CD38 antibody-dependent cell-mediated cytotoxicity inH929 cells, H929 cells were stained with carboxyfluorescein succinimidylester (CFSE) and cultured with PBMCs in the presence or absence ofpomalidomide (0.1 μM), Compound B (0.25, 0.5, 1 μM) for 72 h. The cellswere then incubated with an anti-CD38 antibody (0.5 μg/ml) for 3 hours.Cytotoxicity was assessed by flow cytometry. Compound B combined withpomalidomide enhanced ADCC (FIG. 6). Similarly, Compound B aloneenhanced anti-CD38 antibody-induced cytotoxicity in H929.

1. A pharmaceutical combination comprising: a) a histone deacetylase 6(HDAC6) selective inhibitor of Formula I

or a pharmaceutically acceptable salt thereof, wherein, ring B is arylor heteroaryl; R₁ is aryl or heteroaryl, each of which may be optionallysubstituted by OH, halo, or C₁₋₆-alkyl; and R is H or C₁₋₆-alkyl; and b)a CD38 inhibitor, or a pharmaceutically acceptable salt thereof.
 2. Thepharmaceutical combination of claim 1, wherein the combination furthercomprises: c) a compound selected from the group consisting ofthalidomide, pomalidomide, and lenalidomide or an analog thereof, or apharmaceutically acceptable salt thereof.
 3. The pharmaceuticalcombination of claim 1, wherein the compound of Formula I is:

or a pharmaceutically acceptable salt thereof.
 4. The pharmaceuticalcombination of claim 1, wherein the compound of Formula I is:

or a pharmaceutically acceptable salt thereof.
 5. The pharmaceuticalcombination of claim 1, wherein the CD38 inhibitor is a CD38 inhibitoryantibody.
 6. The pharmaceutical combination of claim 1, wherein the CD38inhibitor is selected from the group consisting of daratumumab,isatuximab (SAR650984), and MOR202 (MOR03087), or pharmaceuticallyacceptable salts thereof.
 7. The pharmaceutical combination of claim 5,wherein: the CD38 inhibitory antibody comprises a heavy chain and alight chain, wherein the heavy chain comprises a variable regioncomprising three CDRs comprising amino acid sequences of SEQ ID NOs.: 3,4, and 5, and wherein the light chain comprises a variable regioncomprising three CDRs comprising amino acid sequences of SEQ ID NOs.: 8,9, and
 10. 8. The pharmaceutical combination of claim 5, wherein: theCD38 inhibitory antibody comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO.: 2, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO.:
 7. 9.The pharmaceutical combination of claim 5, wherein: the CD38 inhibitoryantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO.: 1, and a light chain comprising the amino acid sequence ofSEQ ID NO.:
 6. 10. The pharmaceutical combination of claim 1, whereinthe HDAC inhibitor and the CD38 inhibitor are in the same formulation.11. The pharmaceutical combination of claim 10, wherein the combinationfurther comprises one or more pharmaceutically acceptable carriers. 12.The pharmaceutical combination of claim 1, wherein the HDAC inhibitorand the CD38 inhibitor are in separate formulations.
 13. Thepharmaceutical combination of claim 1, wherein the combination furthercomprises dexamethasone. 14-20. (canceled)